Plant proteins

ABSTRACT

Method for controlling the growth of a plant cell or a plant virus within the cell wherein the level and/or activity retinoblastoma protein in that plant cell is increased or decreased by incorporating a recombinant nucleic acid.

[0001] The present invention relates the proteins having biologicalactivity in plant and animal systems, to polynucleotides encoding forthe expression of such proteins, to oligonucleotides for use inidentifying and synthesizing these proteins and polynucleotides, tovectors and cells containing the polynucleotides in recombinant form andto plants and animals comprising these, and to the use of the proteinsand polynucleotides and fragments thereof in the control of plant growthand plant vulnerability to viruses.

[0002] Cell cycle progression is regulated by positive and negativeeffectors. Among the latter, the product of the retinoblastomasusceptibility gene (Rb) controls the passage of mammalian cells throughG1 phase. In mammalian cells, Rb regulates G1/S transit by inhibitingthe function of the E2F family of transcription factors, known tointeract with sequences in the promoter region of genes required forcellular DNA replication (see eg Weinberg, R. A. Cell 81,323 (1995);Nevins, J. R. Science 258,424 (1992)). DNA tumor viruses that infectanimal cells express oncoproteins that interact with the Rb protein viaa LXCXE motif, disrupting Rb-E2F complexes and driving cells intoS-phase (Weinberg ibid; Ludlow, J. W. FASEB J. 7, 866 (1993); Moran, E.FASEB J. 7, 880 (1993); Vousden, K. FASEB J. 7, 872 (1993)).

[0003] The present inventors have shown that efficient replication of aplant geminivirus requires the integrity of an LXCXE amino acid motif inthe viral RepA protein and that RepA can interact with members of thehuman Rb family in yeast (Xie, Q., Suárez-López, P. and Gutiérrez, C.EMBO J. 14, 4073. (1995). The presence of the LXCXE motif in plantD-type cyclins has also been reported (Soni, R., Carmichael, J. P.,Shah, Z. H. and Murray, J. A. H. Plant Cell 7, 85-103 (1995)).

[0004] The present inventors have now identified characteristicsequences of plant Rb proteins and corresponding encodingpolynucleotides for the first time, isolated such a protein andpolynucleotide, and particularly have identified sequences thatdistinguish it from known animal Rb protein sequences. The inventorshave determined that a known DNA sequence from the maize encoding avegetable Rb plant protein and is hereinafter called ZmRb1. ZmRb1 hasbeen demonstrated by the inventors to interact in yeasts with RepA, aplant geminivirus protein containing LXCXE motif essential for itsfunction. The inventors have further determined that geminivirus DNAreplication is reduced in plant cells transfected with plasmids encodingeither ZmRb1 or human p130, a member of the human Rb family.

[0005] Significantly the inventors work suggests that plant and animalcells may share fundamentally similar strategies for growth control, andthus human as well as plant Rb protein such as ZmRb1 will be expected tohave utility in, inter alia, plant therapeutics, diagnostics, growthcontrol or investigations and many such plant proteins will have similarutility in animals.

[0006] In a first aspect of the present invention there is provided theuse of retinoblastoma protein in controlling the growth of plant cellsand/or plant viruses. Particularly, the present invention providescontrol of viral infection and/or growth in plant cells wherein thevirus requires the integrity of an LXCXE amino acid motif in one of itsproteins, particularly, e. g., in the viral RepA protein, for normalreproduction. Particular plant viruses so controlled are Geminiviruses.

[0007] A preferred method of control using such proteins involvesapplying these to the plant cell, either directly or by introduction ofDNA or RNA encoding for their expression into the plant cell which it isdesired to treat. By over expressing the retinoblastoma protein, orexpressing an Rb protein or peptide fragment thereof that interacts withthe LXCXE motif of the virus but does not affect the normal functioningof the cell, it is possible to inhibit normal virus growth and thus alsoto produce infection spreading from that cell to its neighbours.

[0008] Alternatively, by means of introducing anti-sense DNA or RNA inplant cells in vectors form that contain the necessary promoters for theDNA or RNA transcription, it will be possible to exploit the well knownanti-sense mechanism in order to inhibit the expression of the Rbprotein, and thus the S-phase. Such plants will be of use, among otheraspects to replicate DNA or RNA until high levels, e.g. in yeasts. Themethods to introduce anti-sense DNA in cells are very well known forthose skilled in the art: see for example “Principles of genemanipulation—An introduction to Genetic Engineering (1994) R. W. Old &S. B. Primrose; Oxford-Blackwell Scientific Publications Fifth Editionp398.

[0009] In a second aspect of the present invention there is providedrecombinant nucleic acid, particularly in the form of DNA or cRNA(mRNA), encoding for expression of Rb protein that is characteristic ofplants. This nucleic acid is characterised by one or more characteristicregions that differ from known animal Rb protein nucleic acid and isexemplified herein by SEQ ID No 1, bases 31-2079.

[0010] The DNA or RNA can have a sequence that contains the degeneratedsubstitution in the nucleotides of the codons in SEQ ID No. 1, and inwhere the RNA the T is U. The most preferred DNA or RNA are capable ofhybridate with the polynucleotide of the SEQ ID No. 1 in conditions oflow stringency, preferably being the hybridization produced inconditions of high stringency.

[0011] The expressions “conditions of low stringency” and “conditions ofhigh stringency” are understood by those skilled, but are convenientlyexemplified in U.S. Pat. No. 5,202,257, Col-9-Col 10. If somemodifications were made to lead to the expression of a protein withdifferent amino acids, preferably of the same kind of the correspondingamino acids to the SEQ ID No 1; that is, are conservative substitutions.Such substitutions are known by those skilled, for example, see U.S.Pat. No. 5,380,712, and it is only contemplated when the protein hasactivity with retinoblastoma protein.

[0012] Preferred DNA or cRNA encodes for a plant Rb protein having A andB pocket sub-domains having between 30% and 75% homology with human Rbprotein, particularly as compared with p130, more preferably from 50% to64% homology. Particularly the plant Rb protein so encoded has the C706amino acid of human Rb conserved. Preferably the spacer sequence betweenthe A and B pockets is not conserved with respect to animal Rb proteins,preferably being less than 50% homologous to the same region as found insuch animal proteins. Most preferably the protein so encoded has 80% ormore homology with that of SEQ NO 2 of the sequence listing attachedhereto, still more preferably 90% or more and most preferably 95% ormore. Particularly provided is recombinant DNA of SEQ ID No 1 bases 31to 2079, or the entire SEQ ID No 1, or corresponding RNAs, encoding formaize cDNA clone encoding ZmRb1 of SQ ID No 2.

[0013] In a third aspect of the present invention there is provided theprotein expressed by the recombinant DNA or RNA of the second aspect,novel proteins derived from such DNA or RNA, and protein derived fromnaturally occurring DNA or RNA by mutagenic means such as use ofmutagenic PCR primers.

[0014] In a fourth aspect there are provided vectors, cells and plantsand animals comprising the recombinant DNA or RNA of correct sense oranti-sense, of the invention.

[0015] In a particularly preferred use of the first aspect there isprovided a method of controlling cell or viral growth comprisingadministering the DNA, RNA or protein of the second or third aspects tothe cell. Such administration may be direct in the case of proteins ormay involve indirect means, such as electroporation of plant seed cellswith DNA or by transformation of cells with expression vectors capableof expressing or over expressing the proteins of the invention orfragments thereof that are capable of inhibiting cell or viral growth.

[0016] Alternatively, the method uses an expression vector capable ofproducing anti-sense RNA of the cDNA of the invention.

[0017] Another one of the specific characteristics of the plants proteinand of the nucleic acids includes a N-terminal domain corresponding insequence to the amino acids 1 to 90 of the SEQ ID No. 2 and anucleotides sequence corresponding to the basis 31 to 300 of the SEQ IDNo. 1. These sequences are characterized by possessing less than 150 andless than 450 units that the animal sequences which possess more than300 amino acids and 900 pairs of more bases.

[0018] The present invention will now be illustrated further byreference to the following non-limiting Examples. Further embodimentsfalling within the scope of the claims attached hereto will occur tothose skilled in the light of these.

FIGURES

[0019]FIG. 1. The sub-figure A shows the relative lengths of the presentZmRb1 protein and the human retinoblastoma proteins. The sub-figure Bshows the alignment of the amino acids sequences of the Pocket A andPocket B of the ZmRb1 with that of the Xenopus, chicken, rat and threehuman protein (Rb, p107 and p130).

[0020]FIG. 2. This figure is a map of the main characteristics of theWDV virus and the pWori vector derived from WDV and the positions of thedeletions and mutations used in order to establish that the LXCXE motifis required for its replication in plants cells.

EXAMPLE 1

[0021] Isolation of DNA and Protein Expressing Clones.

[0022] Total RNA was isolated from maize root and mature leaves bygrinding the material previously frozen in liquid nitrogen essentiallyas described in Soni et al (1995). The major and minor p75ZmRb1 mRNAswere identified by hybridization to a random-primed 32P-labelled PstIinternal fragment (1.4 kb).

[0023] A portion of a maize cDNA library (106 pfu) in 1ZAPII(Stratagene) was screened by subsequent hybridization to 5′-labelledoligonucleotides designed to be complementary to a known EST sequence ofhomologue maize of p130. These oligonucleotides were5′-AATAGACACATCGATCAA/G (M.5 m, nt positions 1411-1438) and5′-GTAATGATACCAACATGG (M.3 c, nt positions 1606-1590)(IsogenBiosciences).

[0024] After the second round of screening, pBluescript SK-(pBS)phagemids from positive clones were isolated by in vivo excision withExAssist helper phage (Stratagene) according to protocols recommended bythe manufacturer. DNA sequencing was carried out using a Sequenase™ Kit(USB).

[0025] The 5′-end of the mRNAs encoding p75ZmRb1 was determined byRACE-PCR. Poly-A+mRNA was purified by chromatography onoligo-dT-cellulose (Amersham). The first strand was synthesized usingoligonucleotide DraI35 (5′-GATTTAAAATCAAGCTCC, nt positions 113-96).After denaturation at 90° C. for 3 min, RNA was eliminated by RNasetreatment, the cDNA recovered and 5′-tailed with terminal transferaseand dATP. Then a PCR fragment was amplified using primer DraI35 and thelinker-primer (50 bp) of the Stratagene cDNA synthesis kit.

[0026] One of the positive clones so produced contained a ˜4 kb insertthat, according to restriction analysis, extended both 5′ and 3′ of theregion contained in the Expressed Sequence Tag used. The nucleotidesequence corresponding to the longest cDNA insert (3747 bp) is shown inSEQ ID No. 1. This ZmRb1 b cDNA contains a single open reading framecapable of encoding a protein of 683 amino acids (predicted Mr 75247,p75ZmRb1) followed by a 1646 bp 3′-untranslated region. Untranslatedregions of similar length have been also found in mammalian Rb cDNAs(Lee, W.-L. et al, Science 235, 1394 (1987); Bernards, R. et al, Proc.Natl. Acad. Sci. USA 86, 6474 (1989)). Northern analysis indicates thatmaize cells derived from both root meristems and mature leaves contain amajor message, ˜2.7±0.2 kb in length. In addition, a minor ˜3.7±0.2 kbmessage also appears. Heterogeneous transcripts have been detected inother species (Destrée, O. H. J. et al, Dev. Biol. 153, 141 (1992)).

[0027] Plasmid pWoriΔΔ was constructed by deleting in pWori most of thesequences encoding WDV proteins (Sanz and Gutierrez, unpublished).Plasmid p35S.Rb1 was constructed by inserting the CaMV 35S promoter(obtained from pWDV3:35SGUS) upstream of the ZmRb1 cDNA in the pBSvector. Plasmid p35S.130 was constructed by introducing the completecoding sequence of human p130 instead of ZmRb1 sequences into p35S.Rb1.Plasmid p35.A+B was constructed by substituting sequences encoding theWDV RepA and RepB ORFs instead of ZmRb1 in p35S.Rb1 plasmid. (See Soni,R. and Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)).

[0028] The sequence around the methionine codon at nucleotide position31 contains a consensus translation start (Kozak, M. J. Mol. Biol. 196,947 (1987)). To determine whether the cDNA contained the full-lengthZmRb1 coding region, the 5′-end of the mRNAs was amplified by RACE-PCRusing an oligonucleotide derived from a region close to the putativeinitiator AUG, which would produce a fragment of ˜150 bp. The resultsare consistent with the ZmRb1 cDNA clone containing the complete codingregion.

[0029] The ZmRb1 protein contains segments homologous to the A and Bsubdomains of the “pocket” that is present in all members of the Rbfamily. These subdomains are separated by a non-conserved spacer. ZmRb1also contains non-conserved N-terminal and C-terminal domains. Overall,ZmRb1 shares ˜28-30% amino acid identity (˜50% similarity) with the Rbfamily members (Hannon, G. J., Demetrick, D. & Beach, D. Genes Dev. 7,2378 (1993); Cobrinik, D., Whyte, P., Peeper, D. S., Jacks, T. &Weinberg, R. A. ibid., p. 2392 (1993). Ewen, M. E., Xing, Y. Lawrence,J. B. and Livingston, D. M. Cell 66, 1155 (1991))(Lee W. L. et al,Science 235, 1394 (1987); Bernards et al, Proc. Natl. Acad. Sci. USA 86,6974 (1989)), with the A and B subdomains exhibiting the highesthomology (˜50-64%). Interestingly, amino acid C706 in human Rb, criticalfor its function (Kaye, F. J., Kratzke R. A., Gerster, J. L. andHorowitz, J. M. Proc. Natl. Acad. Sci. USA 87, 6922 (1990)), is alsoconserved in maize p75ZmRb1.

[0030] Note: The 561-577 amino acids encompass a proline-rich domain.

[0031] ZmRb1 contains 16 consensus sites, SP or TP for phosphorilationby cyclins dependant kinases (CDKS) with one of the 5′-tail of thesub-domain A and several in the C-terminal area which are potentialsites of phosphorilation. A nucleic acid preferred group which encodesproteins in which one or more of these sites are changed or deleted,making the protein more resistant to the phosphorilation and thus, toits functionality, for example linking to E2F or similar. This can beeasily carried out by means of mutagenesis conducted by means of PCR.

EXAMPLE 2

[0032] In Vivo Activity.

[0033] Replication of wheat dwarf geminivirus (WDV) is dependent upon anintact LXCXE motif of the viral RepA protein. This motif can mediateinteraction with a member of the human Rb family, p130, in yeasts.Therefore, the inventors investigated whether p75ZmRb1 could complexwith WDV RepA by using the yeast two-hybrid system (Fields, S. and Song,O. Nature 340, 245-246 (1989)). Yeast cells were co-transformed with aplasmid encoding the fusion GAL4BD-RepA protein and with plasmidsencoding different GAL4AD fusion protein. The GAL4AD-p75ZmRb1 fusioncould also complex with GAL4BD-RepA to allow growth of the recipientyeast cells in the absence of histidine. This interaction was slightlystronger than that seen with the human p130 protein. RepA could alsobind to some extent to a N-terminally truncated form of p75ZmRb1. Therole of the LXCXE motif in RepA-p75ZmRb1 interaction was assessed usinga point mutation in WDV RepA (E198K) which we previously showed todestroy interaction with human p130. Co-transformation of ZmRb1 with aplasmid encoding the fusion GAL4BD-RepA(E198K) indicated that theinteraction between RepA and p75ZmRb1 occurred through the LXCXE motif.

[0034] In this respect, the E198K mutant of WDV RepA behaves similarlyto analogous point mutants of animal virus oncoproteins (Moran, E.,Zerler, B., Harrison, T. M. and Mathews, M. B. Mol. Cell Biol. 6, 3470(1986); Cherington, V. et al., ibid., p. 1380 (1988); Lillie, J. W.,Lowenstein, P. M., Green, M. R. and Green, M. Cell 50, 1091 (1987);DeCarpio, J. A. et al., ibid., p. 275 (1988)).

[0035] Specific interaction between maize p75ZmRb1 and WDV RepA in theyeast two-hybrid system (Fields et al) relied on the ability toreconstitute a functional GAL4 activity from two separated GAL4 fusionproteins containing the DNA binding domain (GAL4BD) and the activationdomain (GAL4AD). Yeast HF7c cells were co-transformed with a plasmidexpressing the GAL4BD-RepA or the GAL4BD-RepA(E198K) fusions and theplasmids expressing the GAL4AD alone (Vec) or fused to human p130, maizep75 (p75ZmRb1) or a 69 amino acids N-terminal deletion of p75(p75ZmRb1-DN). Cells were streaked on plates with or without histidineaccording to the distribution shown in the upper left corner. Theability to grow in the absence of histidine depends on the functionalreconstitution of a GAL4 activity upon interaction of the fusionproteins, since this triggers expression of the HIS3 gene which is underthe control of a GAL4 responsive element. The growth characteristics ofthese yeast co-transformants correlate with the levels ofb-galactosidase activity.

[0036] Procedures for two-hybrid analysis are described in Xie et al(1995). The GAL4AD-ZmRb1 fusions were construed in the pGAD424 vector.

EXAMPLE 3

[0037] In Vivo Activity.

[0038] Geminivirus DNA replication requires the cellular DNA replicationmachinery as well as other S-phase specific factors (Davies, J. W. andStanley, J. Trends Genet. 5, 77 (1989); Lazarowitz, S. Crit. Rev. PlantSci. 11, 327 (1992)). Consistent with this requirement, geminivirusinfection appears to drive non-proliferating cells into S-phase, asindicated by the accumulation of the proliferating cell nuclear antigen(PCNA), a protein which is not normally present in the nuclei ofdifferentiated cells (Nagar, S., Pedersen, T. J., Carrick, K. M.,Hanley-Bowdoin, L. and Robertson, D. Plant Cell 7, 705 (1995)). Theinventors finding that efficient WDV DNA replication requires an intactLXCXE motif in RepA coupled with the discovery of a plant homolog of Rbsupports the model that, as in animal cells, sequestration of plant Rbby viral RepA protein promotes inappropriate entry of infected cellsinto S-phase. Therefore, one way to investigate the function of p75ZmRb1was to measure geminivirus DNA replication in cells transfected with aplasmid bearing the ZmRb1 sequences under a promoter functional in plantcells, an approach analogous to that previously used in human cells(Uzvolgi, E. et al., Cell Growth Diff 2, 297 (1991)). Accumulation ofnewly replicated viral plasmid DNA was impaired in wheat cellstransfected with plasmids expressing p75ZmRb1 or human p130, whenexpression of WDV replication protein(s) is directed wither by the WDVpromoter or by the CaMV 35S promoter.

[0039] Since WDV DNA replication requires an S-phase cellularenvironment, interference with viral DNA replication by p75ZmRb1 andhuman p130 strongly evidences a role for retinoblastoma protein in thecontrol of the G1/S transition in plants. The existence of a plant Rbhomolog implies that despite their ancient divergence, plant and animalcells use, at least in part, similar regulatory proteins and pathwaysfor cell cycle control.

[0040] Two lines of evidences reinforce this model. First, a geneencoding a protein that complements specifically the G1/S, but not theG2/M transition of the budding yeast cdc28 mutant has been identified inalfalfa cells (Hirt, H., Páy, A., Bögre, L., Meskiene, I. andHeberle-Bors, E. Plant J. 4, 61 (1993)). Second, plant homologs ofD-type cyclins have been isolated from Arabidopsis and these, like theirmammalian relatives, contain LXCXE motifs. In concert with plantversions of CDK4 and CDK6, plant D-type cyclins may regulate passagethrough G1 phase by controlling the phosphorylation state of Rb-likeproteins.

[0041] In animal cells, the Rb family has been implicated in tumorsuppression and in the control of differentiation and development. Thus,p75ZmRb1 could also play key regulatory roles at other levels during theplant cell life. One key question that is raised by the existence of Rbhomologs in plant cells in whether, as in animals disruption of the Rbpathway leads to a tumor-prone condition. In this regard, the inventorshave noted that the VirB4 protein encoded by the Ti plasmids of bothAgrobacterium tumefaciens and A. rhyzogenes contains an LXCXE motif.Although the VirB4 protein is required for tumor induction (Hooykas, P.J. J. and Beijersbergen, A. G. M. Annu. Rev. Phytopathol. 32, 157(1994), the function of its LXCXE motif in this context remains to beexamined. Geminivirus infection is not accompanied by tumor developmentin the infected plant, but in some cases an abnormal growth of enactionshas been observed (G. Dafalla and B. Gronenborn, personalcommunication).

[0042] Inhibition of wheat dwarf geminivirus (WDV) DNA replication byZmRb1 or human p130 in cultured wheat cells was carried out as follows.A. Wheat cells were transfected, as indicated, with pWori (Xie et al.1995) alone (0.5 g), a replicating WDV-based plasmid which encodes WDVproteins required for viral DNA replication, and with control plasmidpBS (10 g) or p35S.Rb1 (10 g), which encodes ZmRb1 sequences under thecontrol of the CaMV 35S promoter. Total DNA was purified one and twodays after transfection, equal amounts fractionated in agarose gels andethidium bromide staining and viral pWori DNA identified by Southernhybridization. Plasmid DNA represents exclusively newly-replicatedplasmid DNA since it is fully resistant to DpnI digestion and sensitiveto Mbol. Note that the MboI-digested samples were run for about half ofthe length than the undigested samples. B. To test the effect of humanp130 on WDV DNA replication, wheat cells were co-transfected with pWori(0.5 g) and plasmids pBS (control), p35S.Rb1 or p35S.130 (10 g in eachcase). Replication of the test plasmid (pWori) was analyzed two daysafter transfection and was detected as described in part A usingethidium bromide staining; and Southern hybridization. C. To test theeffect of ZmRb1 or human p130 on WDV DNA replication when expression ofviral proteins was directed by the CaMV 35S promoter, the test plasmidpWoriΔΔ (which does not encode functional WDV replication proteins butreplicates when they are provided by a different plasmid, i. e. pWori)was used. Wheat cells were co-transfected, as indicated, with pWoriΔΔ(0.25 g), pWori (0.25 g), p35S.A+B (6 g), p35S.Rb1 (10 g) and/orp35S.130 (10 g). Replication of the test plasmid (pWoriΔΔ) was analyzed36 hours after transfection and was detected as described in part Ausing ethidium bromide staining; Southern hybridization. Plasmids pWori(M1) and pWoriΔΔ (M2; Sanz and Gutiérrez, unpublished), 100 pg in eachcase, were used as markers. Suspension cultures of wheat cells,transfection by particle bombardment and analysis of viral DNAreplication were carried out as described in (Xie et al. 1995), exceptthat DNA extraction was modified as in (Soni and Murray. Arnal. Biochem.218, 474-476 (1995).

1 19 1 683 PRT Unknown Organism Description of Unknown Organism plant RBprotein 1 Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu CysAsn 1 5 10 15 Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile LeuIle Asn 20 25 30 Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Asp SerThr Asn 35 40 45 Leu Gly His Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser ProThr Lys 50 55 60 Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Pro Leu SerPro Ala 65 70 75 80 Thr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro ValThr Ser Ala 85 90 95 Met Thr Thr Ala Lys Trp Leu Arg Glu Val Ile Ser SerLeu Pro Asp 100 105 110 Lys Pro Ser Ser Lys Leu Gln Gln Phe Leu Ser SerCys Asp Arg Asp 115 120 125 Leu Thr Asn Ala Val Thr Glu Arg Val Ser IleVal Leu Glu Ala Ile 130 135 140 Phe Pro Thr Lys Ser Ser Ala Asn Arg GlyVal Ser Leu Gly Leu Asn 145 150 155 160 Cys Ala Asn Ala Phe Asp Ile ProTrp Ala Glu Ala Arg Lys Val Glu 165 170 175 Ala Ser Lys Leu Tyr Tyr ArgVal Leu Glu Ala Ile Cys Arg Ala Glu 180 185 190 Leu Gln Asn Ser Asn ValAsn Asn Leu Thr Pro Leu Leu Ser Asn Glu 195 200 205 Arg Phe His Arg CysLeu Ile Ala Cys Ser Ala Asp Leu Val Leu Ala 210 215 220 Thr His Lys ThrVal Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr 225 230 235 240 Gly LeuThr Ala Phe Asp Leu Ser Lys Ile Ile Glu Asn Phe Val Arg 245 250 255 HisGlu Glu Thr Leu Pro Arg Glu Leu Lys Arg His Leu Asn Ser Leu 260 265 270Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser Leu 275 280285 Tyr Asn Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile Asn 290295 300 Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Asp Asp Leu Val305 310 315 320 Ser Arg Gln Asn Val Arg Ile Glu Gly Leu Pro Ala Thr ProSer Lys 325 330 335 Lys Arg Ala Ala Gly Pro Asp Asp Asn Ala Asp Pro ArgSer Pro Lys 340 345 350 Arg Ser Cys Asn Glu Ser Arg Asn Thr Val Val GluArg Asn Leu Gln 355 360 365 Thr Pro Pro Pro Lys Gln Ser His Met Val SerThr Ser Leu Lys Ala 370 375 380 Lys Cys His Pro Leu Gln Ser Thr Phe AlaSer Pro Thr Val Cys Asn 385 390 395 400 Pro Val Gly Gly Asn Glu Lys CysAla Asp Val Thr Ile His Ile Phe 405 410 415 Phe Ser Lys Ile Leu Lys LeuAla Ala Ile Arg Ile Arg Asn Leu Cys 420 425 430 Glu Arg Val Gln Cys ValGlu Gln Thr Glu Arg Val Tyr Asn Val Phe 435 440 445 Lys Gln Ile Leu GluGln Gln Thr Thr Leu Phe Phe Asn Arg His Ile 450 455 460 Asp Gln Leu IleLeu Cys Cys Leu Tyr Gly Val Ala Lys Val Cys Gln 465 470 475 480 Leu GluLeu Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala 485 490 495 GlnCys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn 500 505 510Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr 515 520525 Asn Glu Val Phe Val Pro Ala Ala Lys Pro Phe Leu Val Ser Leu Ile 530535 540 Ser Ser Gly Thr His Pro Glu Asp Lys Lys Asn Ala Ser Gly Gln Ile545 550 555 560 Pro Gly Ser Pro Lys Pro Ser Pro Phe Pro Asn Leu Pro AspMet Ser 565 570 575 Pro Lys Lys Val Ser Ala Ser His Asn Val Tyr Val SerPro Leu Arg 580 585 590 Gln Thr Lys Leu Asp Leu Leu Leu Ser Pro Ser SerArg Ser Phe Tyr 595 600 605 Ala Cys Ile Gly Glu Gly Thr His Ala Tyr GlnSer Pro Ser Lys Asp 610 615 620 Leu Ala Ala Ile Asn Ser Arg Leu Asn TyrAsn Gly Arg Lys Val Asn 625 630 635 640 Ser Arg Leu Asn Phe Asp Met ValSer Asp Ser Val Val Ala Gly Ser 645 650 655 Leu Gly Gln Ile Asn Gly GlySer Thr Ser Asp Pro Ala Ala Ala Phe 660 665 670 Ser Pro Leu Ser Lys LysArg Glu Thr Asp Thr 675 680 2 3747 DNA Zea mays 2 gaattcggca cgagcaaaggtctgattgat atggaatgtt tccagtcaaa tttggaaaaa 60 atggagaaac tatgtaattctaatagctgt aaaggggagc ttgattttaa atcaattttg 120 atcaataatg attatattccctatgatgag aactcgacgg gggattccac caatttagga 180 cattcaaagt gtgcctttgaaacattggca tctcccacaa agacaataaa gaacatgctg 240 actgttccta gttctcctttgtcaccagcc accggtggtt cagtcaagat tgtgcaaatg 300 acaccagtaa cttctgccatgacgacagct aagtggcttc gtgaggtgat atcttcattg 360 ccagataagc cttcatctaagcttcagcag tttctgtcat catgcgatag ggatttgaca 420 aatgctgtca cagaaagggtcagcatagtt ttggaagcaa tttttccaac caaatcttct 480 gccaatcggg gtgtatcgttaggtctcaat tgtgcaaatg cctttgacat tccgtgggca 540 gaagccagaa aagtggaggcttccaagttg tactataggg tattagaggc aatctgcaga 600 gcggagttac aaaacagcaatgtaaataat ctaactccat tgctgtcaaa tgagcgtttc 660 caccgatgtt tgattgcatgttcagcggac ttagtattgg cgacacataa gacagtcatc 720 atgatgtttc ctgctgttcttgagagtacc ggtctaactg catttgattt gagcaaaata 780 attgagaact ttgtgagacatgaagagacc ctcccaagag aattgaaaag gcacctaaat 840 tccttagaag aacagcttttggaaagcatg gcatgggaga aaggttcatc attgtataac 900 tcactgattg ttgccaggccatctgttgct tcagaaataa accgccttgg tcttttggct 960 gaaccaatgc catctcttgatgacttagtg tcaaggcaga atgttcgtat cgagggcttg 1020 cctgctacac catctaaaaaacgtgctgct ggtccagatg acaacgctga tcctcgatca 1080 ccaaagagat cgtgcaatgaatctaggaac acagtagtag agcgcaattt gcagacacct 1140 ccacccaagc aaagccacatggtgtcaact agtttgaaag caaaatgcca tccactccag 1200 tccacatttg caagtccaactgtctgtaat cctgttggtg ggaatgaaaa atgtgctgac 1260 gtgacaattc atatattcttttccaagatt ctgaagttgg ctgctattag aataagaaac 1320 ttgtgcgaaa gggttcaatgtgtggaacag acagagcgtg tctataatgt cttcaagcag 1380 attcttgagc aacagacaacattatttttt aatagacaca tcgatcaact tatcctttgc 1440 tgtctttatg gtgttgcaaaggtttgtcaa ttagaactca cattcaggga gatactcaac 1500 aattacaaaa gagaagcacaatgcaagcca gaagtttttt caagtatcta tattgggagt 1560 acgaaccgta atggggtattagtatcgcgc catgttggta tcattacttt ttacaatgag 1620 gtatttgttc cagcagcgaagcctttcctg gtgtcactaa tatcatctgg tactcatcca 1680 gaagacaaga agaatgctagtggccaaatt cctggatcac ccaagccatc tcctttccca 1740 aatttaccag atatgtccccgaagaaagtt tcagcatctc ataatgtata tgtgtctcct 1800 ttgcggcaaa ccaagttggatctactgctg tcaccaagtt ccaggagttt ttatgcatgc 1860 attggtgaag gcacccatgcttatcagagc ccatctaagg atttggctgc tataaatagc 1920 cgcctaaatt ataatggcaggaaagtaaac agtcgattaa atttcgacat ggtgagtgac 1980 tcagtggtag ccggcagtctgggccagata aatggtggtt ctacctcgga tcctgcagct 2040 gcatttagcc ccctttcaaagaagagagag acagatactt gatcaattat aaatggtggc 2100 ctctctcgta tatagctcacagatccgtgc tccgtagcag tctattcttc tgaataagtg 2160 gattaactgg agcgatttaactgtacatgt atgtgttagt gagaagcagc agtttttagg 2220 cagcaaactg tttcaagttagcttttgagc tatcaccatt tctctgctga ttgaacatat 2280 ccgctgtgta gagtgctaatgaatctttag ttttcattgg gctgacataa caaatcttta 2340 tcctagttgg ctggttgttgggaggcattc atcagggtta tatttggttg tcaaaaagta 2400 ctgtacttaa ttcacatctttcacattttt cactagcaat agcagcccca aattgctttc 2460 ctgactagga acatattctttacaggtata agcatgccaa ctctaaacta tatgaatcct 2520 ttttatattc tcatttttaagtacttctct gtttctgcta cttttgtact gtatatttcc 2580 agcttctcca tcagactgatgatcccatat tcagtgtgct gcaagtgatt tgaccatatg 2640 tggcttatcc ttcaggtatgtctcatgttg tgacttcatt gctgattgct tttgtaatgg 2700 tactgttgag ttcatttctggttacaatca gcctttactg ctttatattg ttctactaat 2760 tttggcttgc acagccaggacgattggttt tctgcatcaa tcaatctttt ttaggacaag 2820 atatttttgt atgctacacttcccaaattg caattaatcc agaagtctac cttgttttat 2880 tctattagtt ctcagcaacagtgaatgaat atgaatcagt catgctgata gatgttcatc 2940 tggttattcc aaacaatctgacatcgcatc tctttctgca agtgagatga agaaaacctg 3000 aaatgctatc accatttaaaacattggctt ctggaagttc aggtgattag caggagacgt 3060 tctgacattg ccattgacatgtacggtagt gatggcagga gacgttctta aacagcagct 3120 gctccttcag cttgtaatgtctgattgtat tgaccaagag catccacctt gccttatggt 3180 actaactgaa tgagctggtgacgctgactc atctgcataa tggcagatgc ttaaccatct 3240 ttaggagctc atgtcatgattccagctgca ccgtgtcaaa tgtgaaggcc ctgcaaggct 3300 ttccaggccg caccaatcctgcttgcttct tgaagataca tatggtgcca cctaaataaa 3360 agctgtttct ggttatgtctgtccttgaca tgtcaacaga ttagtgttgg gttgcagtca 3420 tgtggtgttt aagtcttggagaaggcgaga agtcattgct gccagcattg tgatcgtcag 3480 gcacagaagt actcaaaagtgagagctact tgttgcgagc aaacggaggg cgatataggt 3540 tgatagccaa tttcagttctctatatacaa gcagcggatt ttgtttagag ttagcttttg 3600 agatgcatca tttctttcacatctgattct gtgtgttgta actcggagtc gcgtagaagt 3660 tagaatgcta actgaccttaattttcaccg aataatttgc tagcgttttt cagtatgaaa 3720 tccttgtctt aaaaaaaaaaaaaaaaa 3747 3 19 DNA Artificial Sequence Description of ArtificialSequence Probe 3 aatagacaca tcgatcaag 19 4 18 DNA Artificial SequenceDescription of Artificial Sequence Probe 4 gtaatgatac caacatgg 18 5 18DNA Artificial Sequence Description of Artificial Sequence Primer 5gatttaaaat caagctcc 18 6 199 PRT Zea mays 6 Thr Pro Val Thr Ser Ala MetThr Thr Ala Lys Trp Leu Arg Glu Val 1 5 10 15 Ile Ser Ser Leu Pro AspLys Pro Ser Ser Lys Leu Gln Gln Phe Leu 20 25 30 Ser Ser Cys Asp Arg AspLeu Thr Asn Ala Val Thr Glu Arg Val Ser 35 40 45 Ile Val Leu Glu Ala IlePhe Pro Thr Lys Ser Ser Ala Asn Arg Gly 50 55 60 Val Ser Leu Gly Leu AsnCys Ala Asn Ala Phe Asp Ile Pro Trp Ala 65 70 75 80 Glu Ala Arg Lys ValGlu Ala Ser Lys Leu Tyr Tyr Arg Val Leu Glu 85 90 95 Ala Ile Cys Arg AlaGlu Leu Gln Asn Ser Asn Val Asn Asn Leu Thr 100 105 110 Pro Leu Leu SerAsn Glu Arg Phe His Arg Cys Leu Ile Ala Cys Ser 115 120 125 Ala Asp LeuVal Leu Ala Thr His Lys Thr Val Ile Met Met Phe Pro 130 135 140 Ala ValLeu Glu Ser Thr Gly Leu Thr Ala Phe Asp Leu Ser Lys Ile 145 150 155 160Ile Glu Asn Phe Val Arg His Glu Glu Thr Leu Pro Arg Glu Leu Lys 165 170175 Arg His Leu Asn Ser Leu Glu Glu Gln Leu Leu Glu Ser Met Ala Trp 180185 190 Glu Lys Gly Ser Ser Leu Tyr 195 7 199 PRT Xenopus sp. 7 Thr ProVal Arg Gly Ala Met Asn Thr Val Gln Gln Leu Met Val Thr 1 5 10 15 LeuSer Ser Ala Asn Asp Lys Pro Pro Asp Thr Leu Asp Ser Tyr Phe 20 25 30 SerAsn Cys Thr Val Asn Pro Lys Thr Lys Ile Thr Asp Arg Ile Glu 35 40 45 HisPhe Gly His Val Phe Lys Glu Lys Phe Ala Ser Ser Val Gly Gln 50 55 60 AlaCys Ala Glu Ile Gly Tyr Gln Arg Tyr Lys Leu Gly Val Cys Leu 65 70 75 80Tyr Tyr Arg Val Met Glu Ala Ile Leu Lys Thr Glu Glu Glu Arg Leu 85 90 95Ser Val His Asn Phe Ser Lys Leu Leu Asn Asn Asp Ile Phe His Ile 100 105110 Cys Leu Leu Ala Cys Ala Val Glu Val Val Val Ala Ser Tyr Ala Arg 115120 125 Asn Ala Ser Gln Ala Tyr Cys Ser Ser Gly Thr Asn Leu Ser Phe Pro130 135 140 Trp Ile Leu Arg Ala Phe Glu Leu Lys Ala Phe Asp Phe Tyr LysVal 145 150 155 160 Ile Glu Cys Phe Ile Lys Ala Glu Pro Ser Leu Thr SerAsn Met Ile 165 170 175 Lys Tyr Leu Glu Arg Cys Glu His Gln Ile Met GluCys Leu Ala Trp 180 185 190 Gln Ser Asp Ser Pro Leu Phe 195 8 200 PRTGallus sp. 8 Thr Pro Val Arg Ala Ala Met Asn Thr Ile Gln Gln Leu Met MetIle 1 5 10 15 Leu Asn Ser Ala Thr Asp Lys Pro Ser Asp Thr Leu Ile AlaTyr Phe 20 25 30 Asn Asn Cys Thr Val Asn Pro Glu Asp Ser Ile Leu Lys ArgVal Glu 35 40 45 Cys Leu Gly His Ile Phe Lys Lys Lys Phe Ala Glu Ala ValGly Gln 50 55 60 Gly Cys Ala Glu Ile Gly Ser Gln Arg Tyr Gln Leu Gly ValArg Leu 65 70 75 80 Tyr Tyr Arg Val Met Glu Ser Met Leu Lys Ser Glu GluGlu Arg Leu 85 90 95 Ser Val His Asn Phe Ser Lys Leu Leu Asn Asp Asn IlePhe His Thr 100 105 110 Ser Leu Leu Ala Cys Ala Leu Glu Ile Val Met AlaThr Tyr Gly Arg 115 120 125 Thr Ala Ser Gln Ser Asp Gly Thr Ser Ala GluThr Asp Leu Ser Phe 130 135 140 Pro Trp Ile Leu Asn Val Phe Asp Leu LysAla Phe Asp Phe Tyr Lys 145 150 155 160 Val Ile Glu Ser Phe Ile Lys ValGlu Pro Ser Leu Thr Arg Asp Met 165 170 175 Ile Lys His Leu Glu Arg CysGlu His Arg Ile Met Glu Ser Leu Ala 180 185 190 Trp Gln Ser Asp Ser ProLeu Phe 195 200 9 198 PRT Mus sp. 9 Thr Pro Val Arg Thr Val Met Asn ThrIle Gln Gln Leu Met Val Ile 1 5 10 15 Leu Asn Ser Ala Ser Asp Gln ProSer Glu Asn Leu Ile Ser Tyr Phe 20 25 30 Asn Asn Cys Thr Val Asn Pro LysGlu Asn Ile Leu Lys Arg Val Lys 35 40 45 Asp Val Gly His Ile Phe Lys GluLys Phe Ala Asn Ala Val Gly Gln 50 55 60 Gly Cys Val Asp Ile Gly Val GlnArg Tyr Lys Leu Gly Val Arg Leu 65 70 75 80 Tyr Tyr Arg Val Met Glu SerMet Leu Lys Ser Glu Glu Glu Arg Leu 85 90 95 Ser Ile Gln Asn Phe Ser LysLeu Leu Asn Asp Asn Ile Phe His Met 100 105 110 Ser Leu Leu Ala Cys AlaLeu Glu Val Val Met Ala Thr Tyr Ser Arg 115 120 125 Ser Thr Leu Gln HisLeu Asp Ser Gly Thr Asp Leu Ser Phe Pro Trp 130 135 140 Ile Leu Asn ValLeu Asn Leu Lys Ala Phe Asp Phe Tyr Lys Val Ile 145 150 155 160 Glu SerPhe Ile Lys Val Glu Ala Asn Leu Thr Arg Glu Met Ile Lys 165 170 175 HisLeu Glu Arg Cys Glu His Arg Ile Met Glu Ser Leu Ala Trp Leu 180 185 190Ser Asp Ser Pro Leu Phe 195 10 198 PRT Homo sapiens 10 Thr Pro Val ArgThr Val Met Asn Thr Ile Gln Gln Leu Met Met Ile 1 5 10 15 Leu Asn SerAla Ser Asp Gln Pro Ser Glu Asn Leu Ile Ser Tyr Phe 20 25 30 Asn Asn CysThr Val Asn Pro Lys Glu Ser Ile Leu Lys Arg Val Lys 35 40 45 Asp Ile GlyTyr Ile Phe Lys Glu Lys Phe Ala Lys Ala Val Gly Gln 50 55 60 Gly Cys ValGlu Ile Gly Ser Gln Arg Tyr Lys Leu Gly Val Arg Leu 65 70 75 80 Tyr TyrArg Val Met Glu Ser Met Leu Lys Ser Glu Glu Glu Arg Leu 85 90 95 Ser IleGln Asn Phe Ser Lys Leu Leu Asn Asp Asn Ile Phe His Met 100 105 110 SerLeu Leu Ala Cys Ala Leu Glu Val Val Met Ala Thr Tyr Ser Arg 115 120 125Ser Thr Ser Gln Asn Leu Asp Ser Gly Thr Asp Leu Ser Phe Pro Trp 130 135140 Ile Leu Asn Val Leu Asn Leu Lys Ala Phe Asp Phe Tyr Lys Val Ile 145150 155 160 Glu Ser Phe Ile Lys Ala Glu Gly Asn Leu Thr Arg Glu Met IleLys 165 170 175 His Leu Glu Arg Cys Glu His Arg Ile Met Glu Ser Leu AlaTrp Leu 180 185 190 Ser Asp Ser Pro Leu Phe 195 11 191 PRT Homo sapiens11 Thr Pro Val Ala Ser Ala Thr Gln Ser Val Ser Arg Leu Gln Ser Ile 1 510 15 Val Ala Gly Leu Lys Asn Ala Pro Ser Asp Gln Leu Ile Asn Ile Phe 2025 30 Glu Ser Cys Val Arg Asn Pro Val Glu Asn Ile Met Lys Ile Leu Lys 3540 45 Gly Ile Gly Glu Thr Phe Cys Gln His Tyr Thr Gln Ser Thr Asp Glu 5055 60 Gln Pro Gly Ser His Ile Asp Phe Ala Val Asn Arg Leu Lys Leu Ala 6570 75 80 Glu Ile Leu Tyr Tyr Lys Ile Leu Glu Thr Val Met Val Gln Glu Thr85 90 95 Arg Arg Leu His Gly Met Asp Met Ser Val Leu Leu Glu Gln Asp Ile100 105 110 Phe His Arg Ser Leu Met Ala Cys Cys Leu Glu Ile Val Leu PheAla 115 120 125 Tyr Ser Ser Pro Arg Thr Phe Pro Trp Ile Ile Glu Val LeuAsn Leu 130 135 140 Gln Pro Phe Tyr Phe Tyr Lys Val Ile Glu Val Val IleArg Ser Glu 145 150 155 160 Glu Gly Leu Ser Arg Asp Met Val Lys His LeuAsn Ser Ile Glu Glu 165 170 175 Gln Ile Leu Glu Ser Leu Ala Trp Ser HisAsp Ser Ala Leu Trp 180 185 190 12 200 PRT Homo sapiens 12 Thr Pro ValSer Thr Ala Thr His Ser Leu Ser Arg Leu His Thr Met 1 5 10 15 Leu ThrGly Leu Arg Asn Ala Pro Ser Glu Lys Leu Glu Gln Ile Leu 20 25 30 Arg ThrCys Ser Arg Asp Pro Thr Gln Ala Ile Ala Asn Arg Leu Lys 35 40 45 Glu MetGln Ala Ile Ala Asn Arg Leu Lys Glu Met Phe Glu Ile Tyr 50 55 60 Ser GlnHis Phe Gln Pro Asp Glu Asp Phe Ser Asn Cys Ala Lys Glu 65 70 75 80 IleAla Ser Lys His Phe Arg Phe Ala Glu Met Leu Tyr Tyr Arg Val 85 90 95 LeuGlu Ser Val Ile Glu Gln Glu Gln Lys Arg Leu Gly Asp Met Asp 100 105 110Leu Ser Gly Ile Leu Glu Gln Asp Ala Phe His Arg Ser Leu Leu Ala 115 120125 Cys Cys Leu Glu Val Val Thr Phe Ser Tyr Lys Pro Pro Gly Asn Phe 130135 140 Pro Phe Ile Thr Glu Ile Phe Asp Val Pro Leu Tyr His Phe Tyr Lys145 150 155 160 Val Ile Glu Val Phe Ile Arg Ala Glu Asp Gly Leu Cys ArgGlu Val 165 170 175 Val Lys His Leu Asn Gln Ile Glu Glu Gln Ile Leu AspHis Leu Ala 180 185 190 Trp Lys Pro Glu Ser Pro Leu Trp 195 200 13 137PRT Zea mays 13 Asn Glu Lys Cys Ala Asp Val Thr Ile His Ile Phe Phe SerLys Ile 1 5 10 15 Leu Lys Leu Ala Ala Ile Arg Ile Arg Asn Leu Cys GluArg Val Gln 20 25 30 Cys Val Glu Gln Thr Glu Arg Val Tyr Asn Val Phe LysGln Ile Leu 35 40 45 Glu Gln Gln Thr Thr Leu Phe Phe Asn Arg His Ile AspGln Leu Ile 50 55 60 Leu Cys Cys Leu Tyr Gly Val Ala Lys Val Cys Gln LeuGlu Leu Thr 65 70 75 80 Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu AlaGln Cys Lys Pro 85 90 95 Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr AsnArg Asn Gly Val 100 105 110 Leu Val Ser Arg His Val Gly Ile Ile Thr PheTyr Asn Glu Val Phe 115 120 125 Val Pro Ala Ala Lys Pro Phe Leu Val 130135 14 129 PRT Xenopus sp. 14 Gln Gln Lys Ser Thr Ser Leu Ser Leu PheTyr Lys Lys Val Tyr Leu 1 5 10 15 Leu Ala Tyr Lys Arg Leu Ser Ser LeuCys Ser Ser Leu Leu Ser Asp 20 25 30 His Pro Glu Leu Glu Gln Val Ile TrpThr Leu Leu Gln His Thr Leu 35 40 45 Gln Gln Glu Tyr Glu Leu Met Arg AspArg His Leu Asp Gln Ile Met 50 55 60 Met Cys Ser Met Tyr Gly Ile Cys LysAla Lys Asn Ile Asp Leu Arg 65 70 75 80 Phe Lys Thr Ile Val Thr Ala TyrLys Gly Leu Thr Asn Thr Asn Gln 85 90 95 Glu Thr Phe Lys His Val Leu IleArg Asp Gly Gln His Asp Ser Ile 100 105 110 Ile Val Phe Tyr Asn Leu ValPhe Met Gln Lys Leu Lys Ser His Ile 115 120 125 Leu 15 131 PRT Gallussp. 15 Gln Lys Pro Gln Lys Ser Thr Ser Leu Ser Leu Phe Tyr Lys Lys Val 15 10 15 Phe Arg Leu Ala Tyr Leu Arg Leu His Thr Leu Phe Phe Arg Leu Leu20 25 30 Ser Glu His Pro Asp Leu Glu Pro Leu Ile Trp Thr Leu Phe Gln His35 40 45 Thr Leu Gln Asn Glu Ser Glu Leu Met Arg Asp Arg His Leu Asp Gln50 55 60 Ile Met Met Cys Ser Met Tyr Gly Ile Cys Lys Val Lys Asn Val Asp65 70 75 80 Leu Arg Phe Lys Thr Ile Val Ser Ala Tyr Lys Glu Leu Pro AsnThr 85 90 95 Asn Gln Glu Thr Phe Lys Arg Val Leu Ile Arg Glu Glu Gln TyrAsp 100 105 110 Ser Ile Ile Val Phe Tyr Asn Leu Val Phe Met Gln Lys LeuLys Thr 115 120 125 Asn Ile Leu 130 16 131 PRT Mus sp. 16 Gln Lys ProLeu Lys Ser Thr Ser Leu Ala Leu Phe Tyr Lys Lys Val 1 5 10 15 Tyr ArgLeu Ala Tyr Leu Arg Leu Asn Thr Leu Cys Ala Arg Leu Leu 20 25 30 Ser AspHis Pro Glu Leu Glu His Ile Ile Trp Thr Leu Phe Gln His 35 40 45 Thr LeuGln Asn Glu Tyr Glu Leu Met Arg Asp Arg His Leu Asp Gln 50 55 60 Ile MetMet Cys Ser Met Tyr Gly Ile Cys Lys Val Lys Asn Ile Asp 65 70 75 80 LeuLys Phe Lys Ile Ile Val Thr Ala Tyr Lys Asp Leu Pro His Ala 85 90 95 AlaGln Glu Thr Phe Lys Arg Val Leu Ile Arg Glu Glu Glu Phe Asp 100 105 110Ser Ile Ile Val Phe Tyr Asn Ser Val Phe Met Gln Arg Leu Lys Thr 115 120125 Asn Ile Leu 130 17 130 PRT Homo sapiens 17 Gln Lys Pro Leu Lys SerThr Ser Leu Ser Leu Phe Tyr Lys Lys Val 1 5 10 15 Tyr Arg Leu Ala TyrLeu Arg Asn Thr Leu Cys Glu Arg Leu Leu Ser 20 25 30 Glu His Pro Glu LeuGlu His Ile Ile Trp Thr Leu Phe Gln His Thr 35 40 45 Leu Gln Asn Glu TyrGlu Leu Met Arg Asp Ala His Leu Asp Gln Ile 50 55 60 Met Met Cys Ser MetTyr Gly Ile Cys Lys Val Lys Asn Ile Asp Leu 65 70 75 80 Lys Phe Lys IleIle Val Thr Ala Tyr Lys Asp Leu Pro His Ala Val 85 90 95 Gln Glu Thr PheLys Arg Val Leu Ile Lys Glu Glu Glu Tyr Asp Ser 100 105 110 Ile Ile ValPhe Tyr Asn Ser Val Phe Met Gln Arg Leu Lys Thr Asn 115 120 125 Ile Leu130 18 166 PRT Homo sapiens 18 Asn Arg Pro Lys Arg Thr Gly Ser Leu AlaLeu Phe Tyr Arg Lys Val 1 5 10 15 Tyr His Leu Ala Ser Val Arg Leu ArgAsp Leu Cys Leu Lys Leu Asp 20 25 30 Val Ser Asn Glu Leu Arg Arg Lys IleTrp Thr Cys Phe Glu Phe Thr 35 40 45 Leu Val His Cys Pro Asp Leu Met LysAsp Arg His Leu Asp Gln Leu 50 55 60 Leu Leu Cys Ala Phe Tyr Ile Met AlaLys Val Thr Lys Glu Glu Arg 65 70 75 80 Thr Phe Gln Glu Ile Met Lys SerTyr Arg Asn Gln Pro Gln Ala Asn 85 90 95 Ser His Val Tyr Arg Ser Val LeuLeu Lys Ser Ile Pro Arg Glu Val 100 105 110 Val Ala Tyr Asn Lys Asn IleAsn Asp Asp Phe Glu Met Ile Asp Cys 115 120 125 Asp Leu Glu Asp Ala ThrLys Thr Pro Asp Cys Ser Ser Gly Pro Val 130 135 140 Lys Glu Glu Arg SerAsp Leu Ile Lys Phe Tyr Asn Thr Ile Tyr Gly 145 150 155 160 Arg Val SerPhe Ala Leu 165 19 195 PRT Homo sapiens 19 Asn Arg Pro Arg Lys Thr SerSer Leu Ser Leu Phe Phe Arg Lys Val 1 5 10 15 Tyr His Leu Ala Ala ValArg Leu Arg Asp Leu Cys Ala Lys Leu Asp 20 25 30 Ile Ser Asp Glu Leu ArgLys Lys Ile Trp Thr Cys Phe Glu Phe Ser 35 40 45 Ile Ile Gln Cys Pro GluLeu Met Met Asp Arg His Leu Asp Gln Leu 50 55 60 Leu Met Cys Ala Ile TyrVal Met Ala Lys Val Thr Lys Glu Asp Lys 65 70 75 80 Ser Phe Gln Asn IleMet Arg Cys Tyr Arg Thr Gln Pro Gln Ala Arg 85 90 95 Ser Gln Val Tyr ArgSer Val Leu Ile Lys Gly Lys Arg Lys Arg Arg 100 105 110 Asn Ser Gly SerSer Asp Ser Arg Ser His Gln Asn Ser Pro Thr Glu 115 120 125 Leu Asn LysAsp Arg Thr Ser Arg Asp Ser Ser Pro Val Met Arg Ser 130 135 140 Ser SerThr Leu Pro Val Pro Gln Pro Ser Ser Ala Ala Pro Thr Pro 145 150 155 160Thr Arg Leu Thr Gly Ala Asn Ser Asp Met Glu Glu Glu Glu Arg Gly 165 170175 Asp Leu Ile Gln Phe Tyr Asn Asn Ile Tyr Ile Lys Gln Ile Lys Thr 180185 190 Phe Ala Met 195

1. A method of controlling the growth of a plant cell or a plant virus within that cell comprising increasing or decreasing the level and/or activity of retinoblastoma protein in that plant cell by incorporation therein of a recombinant nucleic acid.
 2. A method as claimed in claim 1 characterised in that the nucleic acid is such as to increase or inhibit expression of a retinoblastoma protein in the cell.
 3. A method as claimed in claim 1 characterised in that the nucleic acid is such as to express a retinoblastoma protein or peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of the cell.
 4. A method as claimed in claim 3 characterised in that the retinoblastoma protein has been rendered resistant to phosphorylation by cyclin dependent kinases by change or deletion of one or more consensus SP or TP sites found in the SEQ ID No.
 2. 5. A method as claimed in claim 2 characterised in that the DNA or RNA is antisense to retinoblastoma protein encoding DNA or RNA and inhibits retinoblastoma protein expression.
 6. A method of transforming a plant cell such that it is directed into the S shape of the cell cycle comprising introducing a nucleic acid encoding antisense RNA to a plant retinoblastoma protein.
 7. Recombinant nucleic acid encoding for expression of a retinoblastoma protein characterised in that the retinoblastoma protein has an amino acid sequence having 80% or more homology with that of SEQ No. 2 of the sequence listing attached hereto.
 8. Recombinant nucleic acid as claimed in claim 7 characterised in that it comprises SEQ ID no. 1, bases 31-207, sequences only having degenerate substitutions thereof or sequences capable of hybridizing with a polynucleotide of SEQ ID No. 1 under conditions of high stringency.
 9. Recombinant nucleic acid as claimed claim 7 or 8 characterised in that it encodes for a retinoblastoma protein conservatively substituted with respect to SEQ ID No.
 2. 10. Recombinant nucleic acid characterised in that it comprises antisense DNA or RNA to a plant retinoblastoma protein.
 11. Recombinant nucleic acid as claimed in claim 10 characterised in that it comprises antisense DNA or RNA to a plant retinoblastoma protein comprising SEQ ID No. 2 or a sequence having at least 80% homology thereto.
 12. Recombinant nucleic acid as claimed in claim 10 or 11 characterised in that it comprises antisense DNA or RNA to that of SEQ ID No. 1 or a sequence having at least 80% homology thereto.
 13. Recombinant nucleic acid characterised in that it encodes for a retinoblastoma protein or a peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of a plant cell.
 14. Recombinant nucleic acid as claimed in claim 13 characterised in that it encodes for a plant retinoblastoma protein in which one or more consensus SP or TP sites found in the SEQ ID No. 2 have been changed or deleted.
 15. A protein produced by the expression of a recombinant DNA or RNA as claimed in any one of claims 7 to 9, 13 and
 14. 16. A protein as claimed in claim 15 characterised in that one or more consensus SP or TP sites found in the SEQ ID No. 2 have been changed or deleted.
 17. A recombinant vector characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 7 to 9, 13 and
 14. 18. A plant cell characterised in that it comprises a recombinant nucleic acid encoding for expression of a retinoblastoma protein.
 19. A plant cell as claimed in claim 18 characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 7 to 9, 13 and
 14. 20. A plant cell as claimed in claim 18 or 19 characterised in that it expresses a retinoblastoma protein from said nucleic acid.
 21. A transgenic plant characterised in that it comprises a cell as claimed in any one of claims 18 to
 20. 